Snake bite is a serious global public health problem, especially in tropical and subtropical countries (Chippaux 1998). It is estimated that over 5 million snake bite cases occur worldwide each year, of which 2.6 million cause envenomation, and about 125,000 of these result in death (Chippaux 2006). Naja kaouthia (Thai cobra), a member of the Elapidae family, is one of the most venomous and dangerous snakes of Thailand, causing the highest mortality and morbidity due to snake envenomation (Viravan et al. 1986).
Envenomation by N. kaouthia is usually manifested by neurotoxicity and extensive local tissue necrosis (Viravan et al. 1986). The most toxic component of N. kaouthia venom is α-cobratoxin, a low molecular weight (7.8 kDa) post-synaptic α-neurotoxin (Karlsson 1979 cited in Pratanaphon et al. 1997). α-Cobratoxin binds with high affinity and specificity to the nicotinic acetylcholine receptors (nAChRs) on post-synaptic membranes of skeletal muscles, thereby preventing the access of ACh to the receptor's binding pocket (Bourne et al. 2005). Consequently, neuromuscular transmission is blocked and symptoms of muscle flaccid paralysis result. Lethality is generally a result of respiratory failure (Minton 1990). The venom of N. kaouthia also contains several cytotoxins that exhibit cytotoxic activities on many cell types (Inoue et al. 1987). Phospholipases A2 and metalloproteinases are the main components of the venom responsible for local tissue necrosis (Gutierrez et al. 2000).
Antivenoms are currently the only specific treatment for snake bites. Conventional antivenoms are prepared by hyper-immunizing an animal, generally a horse, with snake venom to generate high affinity antibodies against the foreign snake toxins. Horse serum is collected, and whole IgG molecules (150 kDa) are purified and produced into F(ab′)2 antibody fragments (100 kDa) by pepsin digestion and/or Fab antibody fragments (50 kDa) by papain digestion (Lalloo and Theakston 2003). These antibody fragments can then be administered intravenously to an envenomed patient to neutralize the activity of the snake venom toxins. Systemic envenomation is generally treated efficaciously with antivenom; administration of antivenom rapidly neutralizes neurotoxicity caused by the action of post-synaptic neurotoxins (Warrell 1992 cited in Gutierrez et al. 2006). However, antivenoms are ineffective in treating local effects on tissues near the snake bite because of the rapid activity of the toxins at the local tissue, and the inability of antivenom immunoglobulin fragments to reach and penetrate deep tissues (Gutierrez et al. 1998). Although many survive envenomation, a large number of victims are left with chronic physical disability and psychological sequelae as a result of the cytotoxic components of the snake venom (Viravan et al. 1992).
Aside from conventional IgGs, camels and llamas have evolved unique heavy chain IgG immunoglobulins naturally devoid of light chains and the CH1 domains (Hamers-Casterman et al. 1993). The antigen binding sites of these heavy chain IgGs are composed of a single variable domain (called VHHs), and are the smallest natural antigen binding domain (15 kDa). VHH antibody fragments have several properties that potentially would make them superior candidates for antivenom development over conventional antivenoms. They are relatively non-immunogenic, soluble, stable, and highly tissue penetrable (Arbabi Ghahroudi et al. 1997; Cortez-Retamozo et al. 2002 and 2004; Muruganandam et al., 2002). Owing to their low molecular mass, VHH antibody fragments permeate tissue compartments more readily than conventional antibody fragments (Cortez-Retamozo et al., 2002 and 2004) and, therefore, may protect victims from the tissue-damaging effects of venom toxins. Furthermore, because of their small size and high homology to the human VH3 gene family, VHHs may produce less adverse reactions in patients than conventional antivenoms (Vu at al., 1997). Furthermore, VHH antibody fragments can easily be expressed and purified from E. coli/yeast expression systems, solving the current short supply and high cost crisis of antivenoms (Arbabi Ghahroudi et al. 1997; Frenken et al. 2000).
Recently, three VHHs specific for α-cobratoxin were isolated from a naïve (synthetic) llama phage display library (Stewart et al. 2007). However, the affinities of these VHHs were too low (in uM range) for therapeutic efficacy. Therefore, there is a need in the art to obtain higher affinity VHHs against venom such as snake venom.